Direct comparison of the crystal and solution structure of glucose/xylose isomerase from Streptomyces rubiginosus

Glucose isomerase (D-xylose ketol-isomerase, EC 5.3.1.5.) catalyses the isomerization reaction of glucose and xylose. The small angle X-ray scattering (SAXS) data of glucose/xylose isomerase from Streptomyces rubiginosus were recorded for protein solution using synchrotron radiation. The experimental data were compared with theoretical scattering calculated on the basis of the known crystal structure (PDB code: 1OAD). The radius of gyration measured by SAXS (R(G)=3.30 nm) was almost identical and the maximum dimension in the distance distribution function was by about 2.5 % lower than the corresponding values calculated on the basis of the crystal structure.     

A metal-mediated hydride shift mechanism for xylose isomerase based on the 1.6 A Streptomyces rubiginosus structures with xylitol and D-xylose

The crystal structure of recombinant Streptomyces rubiginosus D-xylose isomerase (D-xylose keto-isomerase, EC 5.3.1.5) solved by the multiple isomorphous replacement technique has been refined to R = 0.16 at 1.64 A resolution. As observed in an earlier study at 4.0 A (Carrell et al., J. Biol. Chem. 259: 3230-3236, 1984), xylose isomerase is a tetramer composed of four identical subunits. The monomer consists of an eight-stranded parallel beta-barrel surrounded by eight helices with an extended C-terminal tail that provides extensive contacts with a neighboring monomer. The active site pocket is defined by an opening in the barrel whose entrance is lined with hydrophobic residues while the bottom of the pocket consists mainly of glutamate, aspartate, and histidine residues coordinated to two manganese ions. The structures of the enzyme in the presence of MnCl2, the inhibitor xylitol, and the substrate D-xylose in the presence and absence of MnCl2 have also been refined to R = 0.14 at 1.60 A, R = 0.15 at 1.71 A, R = 0.15 at 1.60 A, and R = 0.14 at 1.60 A, respectively. Both the ring oxygen of the cyclic alpha-D-xylose and its C1 hydroxyl are within hydrogen bonding distance of NE2 of His-54 in the structure crystallized in the presence of D-xylose. Both the inhibitor, xylitol, and the extended form of the substrate, D-xylose, bind such that the C2 and C4 OH groups interact with one of the two divalent cations found in the active site and the C1 OH with the other cation. The remainder of the OH groups hydrogen bond with neighboring amino acid side chains. A detailed mechanism for D-xylose isomerase is proposed. Upon binding of cyclic alpha-D-xylose to xylose isomerase, His-54 acts as the catalytic base in a ring opening reaction. The ring opening step is followed by binding of D-xylose, involving two divalent cations, in an extended conformation. The isomerization of D-xylose to D-xylulose involves a metal-mediated 1,2-hydride shift. The final step in the mechanism is a ring closure to produce alpha-D-xylulose. The ring closing is the reverse of the ring opening step. This mechanism accounts for the majority of xylose isomerase's biochemical properties, including (1) the lack of solvent exchange between the 2-position of D-xylose and the 1-pro-R position of D-xylulose, (2) the chemical modification of histidine and lysine, (3) the pH vs. activity profile, and (4) the requirement for two divalent cations in the mechanism.     

Complex organization of the Streptomyces avermitilis genes encoding the avermectin polyketide synthase.

The avermectin (Av) polyketide synthase (PKS) and erythromycin (Er) PKS are encoded by modular repeats of DNA, but the genetic organization of the modules encoding Av PKS is more complex than Er PKS. Sequencing of several related DNA fragments from Streptomyces avermitilis that are part of the Av biosynthetic gene cluster, revealed that they encode parts of large multifunctional PKS proteins. The Av PKS proteins show strong similarity to each other, as well as similarity to Er PKS proteins [Donadio et al., Science 252 (1991) 675-679] and fatty acid synthases. Partial DNA sequencing of the 65-kb region containing all the related sequence elements in the avr genes provides evidence for twelve modular repeats encoding FAS-like domains. The genes encoding the Av PKS are organized as two sets of six modular repeats which are convergently transcribed.     

Identification of Streptomyces coelicolor genes involved in regulation of antibiotic synthesis.

To define genetic elements that regulate antibiotic synthesis, we screened for mutations that visibly blocked synthesis of Streptomyces coelicolor's two pigmented antibiotics and found mutant strains in which all four antibiotics were blocked. The responsible mutations defined two loci, absA and absB. Two additional approaches to defining genes have been taken: isolation of cloned genes with a dominant negative effect on antibiotic synthesis and isolation of genes which, in multicopy, can compensate for specific mutational blocks. These genes apparently function in a global regulatory pathway (or network) for control of antibiotic synthesis.     

Genetic aspects of the organization of legumin genes in pea

We have compared physical and genetic maps of the region around the legJ gene in pea. In this vicinity there are four B-type legumin genes, arranged as two close pairs. The detection of a recombination event within this gene cluster allows the orientation of this group of genes within the surrounding linkage group to be determined. The relationship between physical and genetic distances in this region is discussed, as are the implications of this for relating physical and genetic maps elsewhere in the pea genome.     

Plasmid localization and organization of melamine degradation genes in Rhodococcus sp. strain Mel

Rhodococcus sp. strain Mel was isolated from soil by enrichment and grew in minimal medium with melamine as the sole N source with a doubling time of 3.5 h. Stoichiometry studies showed that all six nitrogen atoms of melamine were assimilated. The genome was sequenced by Roche 454 pyrosequencing to 13× coverage, and a 22.3-kb DNA region was found to contain a homolog to the melamine deaminase gene trzA. Mutagenesis studies showed that the cyanuric acid hydrolase and biuret hydrolase genes were clustered together on a different 17.9-kb contig. Curing and gene transfer studies indicated that 4 of 6 genes required for the complete degradation of melamine were located on an ～265-kb self-transmissible linear plasmid (pMel2), but this plasmid was not required for ammeline deamination. The Rhodococcus sp. strain Mel melamine metabolic pathway genes were located in at least three noncontiguous regions of the genome, and the plasmid-borne genes encoding enzymes for melamine metabolism were likely recently acquired.     

Thioredoxin-thioredoxin reductase system of Streptomyces clavuligerus: sequences, expression, and organization of the genes.

The genes that encode thioredoxin and thioredoxin reductase of Streptomyces clavuligerus were cloned, and their DNA sequences were determined. Previously, we showed that S. clavuligerus possesses a disulfide reductase with broad substrate specificity that biochemically resembles the thioredoxin oxidoreductase system and may play a role in the biosynthesis of beta-lactam antibiotics. It consists consists of two components, a 70-kDa NADPH-dependent flavoprotein disulfide reductase with two identical subunits and a 12-kDa heat-stable protein general disulfide reductant. In this study, we found, by comparative analysis of their predicted amino acid sequences, that the 35-kDa protein is in fact thioredoxin reductase; it shares 48.7% amino acid sequence identity with Escherichia coli thioredoxin reductase, the 12-kDa protein is thioredoxin, and it shares 28 to 56% amino acid sequence identity with other thioredoxins. The streptomycete thioredoxin reductase has the identical cysteine redox-active region--Cys-Ala-Thr-Cys--and essentially the same flavin adenine dinucleotide- and NADPH dinucleotide-binding sites as E. coli thioredoxin reductase and is partially able to accept E. coli thioredoxin as a substrate. The streptomycete thioredoxin has the same cysteine redox-active segment--Trp-Cys-Gly-Pro-Cys--that is present in virtually all eucaryotic and procaryotic thioredoxins. However, in vivo it is unable to donate electrons to E. coli methionine sulfoxide reductase and does not serve as a substrate in vitro for E. coli thioredoxin reductase. The S. clavuligerus thioredoxin (trxA) and thioredoxin reductase (trxB) genes are organized in a cluster. They are transcribed in the same direction and separated by 33 nucleotides. In contrast, the trxA and trxB genes of E. coli, the only other organism in which both genes have been characterized, are physically widely separated.     

Expression of xylA genes encoding xylose isomerases from Escherichia coli and Streptomyces coelicolor in the methylotrophic yeast Hansenula polymorpha

The thermotolerant methylotrophic yeast Hansenula polymorpha is able to ferment xylose to ethanol at high temperatures. H. polymorpha xylose reductase and xylitol dehydrogenase are involved during the first steps of this fermentation. In this article, expression of bacterial xylA genes coding for xylose isomerases from Escherichia coli or Streptomyces coelicolor in the yeast H. polymorpha was shown. The expression was achieved by integration of the xylA genes driven by the promoter of the H. polymorpha glyceraldehyde-3-phosphate dehydrogenase gene ( HpGAP) into the H. polymorpha genome. Expression of the bacterial xylose isomerase genes restored the ability of the H. polymorpha Deltaxyl1 mutant to grow in a medium with xylose as the sole carbon source. This mutant has a deletion of the XYL1 gene encoding xylose reductase and is not able to grow in the xylose medium. The H. polymorpha Deltaxyl1(xylA) transformants displayed xylose isomerase activities, which were near 20% of that of the bacterial host strain. The transformants did not differ from the yeast wild-type strain with respect to ethanol production in xylose medium.     

Role of invariant water molecules and water-mediated ionic interactions in D-xylose isomerase from Streptomyces rubiginosus

The enzyme, D-xylose isomerase (D-xylose keto-isomerase; EC 5.3.1.5) is a soluble enzyme that catalyzes the conversion of the aldo-sugar D-xylose to the keto-sugar D-xylulose. A total of 27 subunits of D-xylose isomerase from Streptomyces rubiginosus were analyzed in order to identify the invariant water molecules and their water-mediated ionic interactions. A total of 70 water molecules were found to be invariant. The structural and/or functional roles of these water molecules have been discussed. These invariant water molecules and their ionic interactions may be involved in maintaining the structural stability of the enzyme D-xylose isomerase. Fifty-eight of the 70 invariant water molecules (83%) have at least one interaction with the main chain polar atom.     

Molecular cloning of tetracycline resistance genes from Streptomyces rimosus in Streptomyces griseus and characterization of the cloned genes.

Two tetracycline resistance genes of Streptomyces rimosus, an oxytetracycline producer, were cloned in Streptomyces griseus by using pOA15 as a vector plasmid. Expression of the cloned genes, designated as tetA and tetB was inducible in S. griseus as well as in the donor strain. The tetracycline resistance directed by tetA and tetB was characterized by examining the uptake of tetracycline and in vitro polyphenylalanine synthesis by the sensitive host and transformants with the resultant hybrid plasmids. Polyphenylalanine synthesis with crude ribosomes and the S150 fraction from S. griseus carrying the tetA plasmid was resistant to tetracycline, and, by a cross-test of ribosomes and S150 fraction coming from both the sensitive host and the resistant transformant, the resistance directed by tetA was revealed to reside mainly in crude ribosomes and slightly in the S150 fraction. However, the resistance in the crude ribosomes disappeared when they were washed with 1 M ammonium chloride. These results suggest that tetA specified the tetracycline resistance of the machinery for protein synthesis not through ribosomal subunits, but via an unidentified cytoplasmic factor. In contrast, S. griseus carrying the tetB plasmid accumulated less intracellular tetracycline than did the host, and the protein synthesis by reconstituting the ribosomes and S150 fraction was sensitive to the drug. Therefore, it is conceivable that tetB coded a tetracycline resistance determinant responsible for the reduced accumulation of tetracycline.     

Genetic recombination in Streptomyces griseus.

Low-frequency (10(-6)) genetic recombination was observed in a cephamycin-producing strain of Streptomyces griseus. The recombinants were predominantly heteroclones. Heteroclone analysis was performed involving four heteroclones of one cross. In 100 mutants correlation was found between the type of auxotrophy and the level of antibiotic activity. A cross of this strain with a streptomycin-producing strain of S. griesus is described.